MUTAGENIC EFFECTS PHOTOINDUCED IN MAMMALIAN CELLS IN VITRO BY TWO MONOFUNCTIONAL PYRIDOPSORALENS

Abstract— The photobiological activity of the two monofunctional pyridopsoralens pyrido (3,4-c) psoralen (PyPs) and 7-methyl pyrido (3,4-c) psoralen (MePyPs) was studied in mammalian cells in vitro taking 8-methoxypsoralen (8-MOP) as a reference compound.
In the presence of 365-nm irradiation (UVA) MePyPs was found to be more effective than 8-MOP in terms of DNA photobinding capacity and inhibition of cell cloning ability in Chinese hamsterV–79 cells. As a function of UVA dose and of the number of total photoadducts induced MePyPs produced a higher frequency of 6-thioguanine resistant mutants than 8-MOP. PyPs showed an intermediate response for cell killing and mutation induction. At equal cytotoxic levels both monofunctional pyridopsoralens exhibited the same mutagenic activity as the Afunctional furocoumarin 8-MOP.
The antiproliferative effect being taken as indicative for an efficient photochemotherapeutic activity against psoriasis, the inhibition of cloning capacity induced by MePyPs plus UVA was studied in parallel on human skin fibroblasts. Such cells were more sensitive to 8-MOP photoadditions thanV–79 cells and even more so to MePyPs photoadditions. Data obtained on the rate of DNA semi conservative synthesis on both cell lines following treatments with the two compounds are in line with these observations.     

New aspects of the repair and genotoxicity of psoralen photoinduced lesions in DNA.

Several approaches are described aiming at a better understanding of the genotoxicity of psoralen photoinduced lesions in DNA. Psoralens can photoinduce different types of photolesions including 3,4- and 4',5'-monoadducts and interstrand cross-links, oxidative damage (in the case of 3-carbethoxypsoralen (3-CPs)) and even pyrimidine dimers (in the case of 7-methylpyrido(3,4-c)psoralen (MePyPs)). The characterization and detection of different types of lesions has been essential for the analysis of their possible contributions to genotoxicity. For example, oxidative damage photoinduced by 3-CPs can be detected by the formamidopyrimidine glycosylase (FPG) protein. Furthermore, it is shown how the presence of MePyPs induced monoadducts may interfere with the photoreactivation of concomitantly induced pyrimidine dimers, how the ratio of monoadducts and interstrand cross-links (CL) affects the occurrence of double-strand breaks during the repair of photolesions and genotoxicity. In vitro treatment of yeast plasmids, followed by transformation, also indicates that the repair of photoadducts on exogenous DNA differs for 8-methoxy-psoralen (8-MOP) induced mono- and diadducts and for monoadducts alone. The recombinational rad52 dependent pathway is not needed for the repair of 8-MOP induced monoadducts. The results obtained suggest that the genotoxic effects of psoralens are conditioned by the nature, number, ratio and sequence distribution of the photolesions induced in DNA.     

A NEW MONOFUNCTIONAL PYRIDOPSORALEN: PHOTOREACTIVITY and REPAIR IN YEAST

Abstract— The newly synthesized derivative of psoralen, the pyrido (3,4-c) 7 methylpsoralen (MePyPs), acts in combination with 365 nm ultraviolet as a monofunctional agent on yeast DNA. In vivo, its photoaffinity for DNA is much higher than that of the bifunctional agent, 8-methoxypsoralen (8-MOP). The MePyPs photo-induced monoadducts are almost completely removed from wild type cells DNA as efficiently as 8-MOP photo-induced adducts during post-treatment incubation. This process is blocked in excision-repair defective mutants (6 to 10% residual excision in radl- Δ or rad2- Δ ). For an equal number of photoinduced lesions, the DNA single strand breaks which are produced concomitantly to MePyPs or 8-MOP photoadducts excision are rapidly rejoined in the case of 8-MOP whereas they are only partly resealed for the MePyPs treatment. The high photo-toxicity of MePyPs, a promising agent for photo-chemotherapeutic use, is explained in terms of the high photoaffinity for DNA.     

Modulation of base excision repair alters cellular sensitivity to UVA1 but not to UVB1

Oxidative DNA damage has been implicated in some of the biological properties of UVA but so far not in the acute photosensitivity or cellular sensitivity. In contrast to pyrimidine dimers, oxidative DNA damage is predominantly processed by base excision repair (BER). In order to further clarify the role of oxidative DNA damage and its repair in the acute cellular response to UV light, we studied UVA1 and UVB sensitivities in three different cell model systems with modified BER. 8-Oxoguanine-DNA-glycosylase 1-/- (OGG1-/-) mouse embryonal fibroblasts and human fibroblasts in which BER was inhibited by incubation with methoxyamine were hypersensitive to UVA1, in particular to low doses. This hypersensitivity could be partially corrected by reexpression of OGG1 in OGG1-/- cells. The Chinese hamster ovary (CHO) cells with upregulated AP-endonuclease 1 exhibited reduced UVA1 sensitivity. UVB sensitivity was not altered in any of the cell models. These results indicate that DNA damage, in particular oxidative DNA damage, contributes to cellular UVA1 sensitivity and underline a pivotal role of its repair in the cellular responses to UVA1.     

INHIBITION OF TRANSIENT GENE EXPRESSION IN CHINESE, HAMSTER OVARY CELLS BY TRIPLET-SENSITIZED UV-B IRRADIATION OF TRANSFECTED DNA

The biological effectiveness of thymine-thymine cyclobutane dimers specifically induced by photosensitized ultraviolet-B irradiation was analyzed by host-cell reactivation of triplet-sensitized, UV-B irradiated plasmid pRSV beta gal DNA transfected into normal and repair-deficient Chinese hamster ovary cells. For comparison, pRSV beta gal DNA was also UV-C irradiated and transfected into the same cell lines. Ultraviolet endonuclease-sensitive site induction was determined after UV-C irradiation or acetophenone-sensitized UV-B irradiation of plasmid pRSV beta gal DNA. These data were used to calculate the number of cyclobutane pyrimidine dimers required to inactivate expression of the lacZ reporter gene in each irradiation condition. Transfection with UV-C-irradiated plasmid DNA resulted in a significantly greater reduction of reporter gene expression than did transfection with acetophenone-sensitized UV-B-irradiated pRSV beta gal DNA at equivalent induction of enzyme-sensitive sites. Since only a fraction of the inhibition could be accounted for by noncyclobutane dimer photoproducts, these results suggest that cytosine-containing pyrimidine cyclobutane dimers may be more effective than thymine-thymine dimers in inhibiting transient gene expression as measured in such host-cell reactivation experiments in mammalian cells.     

INACTIVATION OF NUCLEOTIDE EXCISION REPAIR IN CHINESE HAMSTER OVARY CELLS BY EXPOSURE TO AN ALKYLATING AGENT

Abstract— Unscheduled DNA synthesis and excision of pyrimidine dimers in Chinese hamster ovary cells irradiated with UV light were inhibited by prior exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)(1–10 μ M ) Although the pathways for excision of pyrimidine dimers and alkylation damage are known to differ, alkylations from MNNG exposure appear to have a direct effect on the nucleotide excision repair system. These results indicate that the method of exposing cells to two DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.     

STUDY OF PYRIMIDINE DIMERS IN MAMMALIAN CELLS SURVIVING LOW DOSES OF ULTRAVIOLET RADIATION*

Abstract— Ultraviolet-induced pyrimidine dimers were not found to be excided from the DNA of Chinese hamster cells in small oligounucleotides. At doses whereby many cells survive the radiation, the dimers were still associates with the large polynucleotides even after 48 hr of postirradiation incubation.     

INDUCTION OF DNA–PROTEIN CROSS-LINKING IN CHINESE HAMSTER CELLS BY MONOCHROMATIC 365 AND 405 NM ULTRAVIOLET LIGHT

Abstract— The survival, the induction of DNA-protein cross-linking, and the number of T4-endonuclease sensitive sites were measured in Chinese hamster cells that had been irradiated with 365 and 405 nm monochromatic light. The survival measurements show that cells are somewhat less sensitive to 405 nm light than to 365 nm light. The difference is expressed predominantly in the shoulder widths of the survival curves, whereas the slopes of the two curves are about the same. Induction of pyrimidine dimers, as indicated by the number of endonuclease-sensitive sites, after exposures that produce about 10% survival is very low at 365 nm (˜ 4 endonuclease sites per 2 × 10⁸ daltons), while no dimers are detected at 405 nm. In contrast, DNA-protein cross-links are induced rather effectively at either wavelength even after exposures that result in a relatively high survival (60-20%). Our measurements support the conclusion that lethality in mammalian cells after irradiations with 365 or 405 nm light is caused by a nondimer damage, possibly DNA-protein cross-links.     

LOCAL SUPPRESSION OF CONTACT HYPERSENSITIVITY IN MICE BY A MONOFUNCTIONAL PSORALEN PLUS UVA RADIATION

Monofunctional psoralens, plus UVA radiation are not erythemogenic and are less mutagenic than bifunctional psoralens plus UVA radiation. Thus, they have received considerable attention in recent years as potential therapeutic agents for various skin diseases. The purpose of this study was to examine the immunologic side effects following treatment of mice with a monofunctional psoralen plus UVA radiation. We report that angelicin plus UVA radiation suppressed the induction of contact hypersensitivity to dinitrofluorobenzene. This decreased immune response was associated with the presence of splenic suppressor cells that transferred suppression to normal recipients. Treatment with angelicin and UVA radiation also decreased the number of Thy-1+ and Ia+ dendritic epidermal cells in the treated site. We conclude that although this monofunctional psoralen is not phototoxic, it has immunosuppressive activity in mice.     

DIFFERENT EFFECTS OF RAD GENES OF SACCHAROMYCES CEREVISIAE ON INCISIONS OF INTERSTRAND CROSSLINKS AND MONOADDUCTS IN DNA INDUCED BY PSORALEN PLUS NEAR UV LIGHT TREATMENT

Abstract— The RAD1, RAD2, RAD3 and RAD4 genes of Saccharomyces cerevisiae are required for incising DNA containing UV induced pyrimidine dimers or psoralen plus 360 nm light induced interstrand crosslinks. We have now determined if these genes are also required for incising DNA at psoralen plus 360 nm light induced monoadducts. For distinguishing between incision breaks occurring at crosslinks and at monoadducts. we have used the cdc9-2 mutant, defective in DNA ligase activity at the restrictive temperature, and the radl-2 cdc9-2, rad2-5 cdc9-2 , rad3-2 cdc9-2 and rad4-4 cdc9-2 double mutant combinations. We conclude that the radl, rad2 , and rad4 mutants are defective in incising DNA both at crosslinks and monoadducts, whereas the rad3 mutant is proficient in incising DNA at monoadducts but not at crosslinks.     

EFFECT OF MOLECULAR STRUCTURE ON THE PHOTOPHYSICAL PROPERTIES, THE PHOTOREACTIVITY WITH DNA AND THE PHOTOBIOLOGICAL ACTIVITY OF MONOFUNCTIONAL PYRIDOPSORALENS

Abstract— 7-Methyl-pyrido[4,3-c]psoralen (2N-MePyPs) has been synthesized in order to investigate the possible effect of the position of the pyridine-nitrogen atom on the photoreactivity towards DNA and the photobiological activity of pyridopsoralens, a new family of psoralen derivatives. In comparison to its isomer, 7-methyl-pyrido[3,4-c]psoralen (MePyPs), 2N-MePyPs shows a 2.5 times lower DNA photobinding capacity. Photobiological experiments with diploid yeast ( Saccharomyces cerevisiae ) reveal that this compound differs strikingly from its isomer MePyPs. It has only a weak antiproliferative potential and, per unit dose, a lower capacity than MePyPs for the induction of nuclear genotoxic effects. With respect to these latter features, 2N-MePyPs resembles the monofunctional furocoumarin 3-CPs.     

Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta).

The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible + ultraviolet-A + ultraviolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB. Damage induced by VIS + UVA + UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS + UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.     

INDUCTION OF phr GENE EXPRESSION BY PYRIMIDINE DIMERS IN Escherichia coli

The photoreactivating enzyme (PRE) is concerned with mainly two kinds of light wavelength. The PRE splits UVC (254 nm)-induced pyrimidine dimer by absorbing UVA (320–380 nm) or visible light in its chromophore. The present paper demonstrates that the phr gene expression was efficiently induced in an excision defective strain (uvrA∼) after irradiation by UVC and UVB (290-320 nm), but not by UVA and visible light. In addition, the induced activity was significantly depressed by irradiation with UVA and visible light. Therefore we conclude that the phr gene expression can be induced by pyrimidine dimers.     

Hydrochlorothiazide Enhances UVA‐Induced DNA Damage

The UVA is currently thought to be carcinogenic because, similar to UVB, it induces the formation of cyclobutane pyrimidine dimers (CPDs). Various drugs have been reported to cause photosensitive drug eruptions as an adverse effect. Although the precise mechanism of photosensitive drug eruption remains to be elucidated, it is generally accepted that free radicals and other reactive molecules generated via UV‐irradiated drugs play important roles in the pathogenesis of photosensitive drug eruptions. The waveband of concern for photo‐reactive drugs is UVA‐visible light, but some extend into the UVB region. We tested whether photosensitive drugs could enhance CPD formation after UVA exposure by using isolated DNA in the presence of several reported photosensitive drugs using high‐performance liquid chromatography. We found that the diuretic agent hydrochlorothiazide (HCT) significantly enhanced the production of TT dimers over a wide range of UVA. Furthermore, we investigated whether UVA plus HCT could enhance CPD production in xeroderma pigmentosum model mice defective in nucleotide excision repair. Immunofluorescence studies showed that CPD formation in the skin significantly increased after 365 nm narrow‐band UVA irradiation in the presence of HCT, compared with that in wild‐type mice. HCT could be used with caution because of its enhancement of UVA‐induced DNA damage.     

Repair of furocoumarin-plus-UVA-induced damage and mutagenic consequences in eukaryotic cells

In the presence of near-UV radiation (UVA) furocoumarins (psoralens) photoinduce defined lesions in DNA, i.e. monoadducts and interstrand crosslinks. Their use in photochemotherapy (psoralen plus UVA (PUVA) treatment) and cosmetics raises questions concerning the repairability of these lesions and their genotoxic consequences. We have analysed the repair of psoralen photoadducts in cultured eukaryotic cells, such as yeast and mammalian cells, for furocoumarins of photochemotherapeutic interest. In yeast, the interaction of repair pathways differs in exogenous (plasmid) and endogenous (chromosomal) DNA. The order of mutagenic activity is 4,5',8-trimethylpsoralen greater than 5-methoxypsoralen greater than 8-methoxypsoralen greater than 7-methylpyrido[3,4-c]psoralen greater than 3-carbethoxypsoralen. The mutagenicity is dependent on psoralen functionality, concentration and bioavailability, maximal UVA dose, wavelength, dose (fluence) rate and presence or absence of chemical filters. It probably involves an inducible component. Chromosome breakage occurs during the repair period after PUVA treatment. It appears that the genotoxic effects of psoralens are produced by a specific arrangement of induced photolesions and the interaction of different repair systems.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

UVB-lnduced DNA Damage and Its Photorepair in Nuclei and Chloroplasts of Spinacia oleracea L.

Ultraviolet-B-induced lesions and their photorepair in nuclear and chloroplast DNA of spinach (Spinacia oleracea L.) leaves were examined with two photoproducts, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidinone photoproducts (6-4PP). These photoproducts were induced both in nuclear and chloroplast DNA by UVB irradiation and could be detected by enzyme-linked immunosorbent assay using their respective monoclonal antibodies. Formation of CPD was greater in nuclear DNA than in chloroplast DNA (about 10 to 7), whereas 6-4PP formation was comparable in both DNA. On subsequent exposure of leaves to blue/UVA after UVB irradiation, photorepair of CPD and 6-4PP occurred in nuclear DNA but not in chloroplast DNA. When isolated chloroplasts were irradiated with UVB, CPD was also induced in their DNA. But photorepair of CPD did not occur in them by subsequent exposure to blue/UVA, suggesting that no photorepair system operates in chloroplasts.     

PHOTOSENSITIZED SPLITTING OF PYRIMIDINE DIMERS BY INDOLE DERIVATIVES AND BY TRYPTOPHAN-CONTAINING OLIGOPEPTIDES AND PROTEINS

Abstract— Indole derivatives including tryptophan can be used as photosensitizers of the splitting of pyrimidine dimers. The reaction can take place in frozen aqueous solutions as well as in fluid medium. Electron transfer from the indole ring to the dimer appears to be involved in the photosensitized reaction. Solvated electrons produced by flash photolysis in the presence of indoles or by pulse radiolysis are also able to split thymine dimers.
The splitting of pyrimidine dimers in DNA can be photosensitized by indole derivatives such as serotonin and by tryptophan-containing oligopeptides. Several methods including fluorescence and nuclear magnetic resonance have been used to show that the indole ring of these oligopeptides is able to stack with bases in nucleic acids. These stacked complexes are involved in the photosensitized reaction.
The splitting of pyrimidine dimers in DNA has also been photosensitized by the protein coded by gene 32 of phage T4 which binds strongly and cooperatively to single-stranded DNA. The mechanism of the splitting reaction as well as the possible use of this reaction to investigate the role of tryptophan residues in the binding of proteins to nucleic acids are discussed.     

OXYGEN-DEPENDENCE OF NEAR UV (365 NM) LETHALITY AND THE INTERACTION OF NEAR UV AND X-RAYS IN TWO MAMMALIAN CELL LINES

Abstract— The colony-forming ability of Chinese hamster cells (V-79) and HeLa cells has been measured after near-ultraviolet (UV) irradiation, predominantly at 365 nm. To avoid the production of toxic photoproducts, cells were irradiated in an inorganic buffer rather than in tissue culture medium. Under these circumstances near-UV lethality was strongly oxygen-dependent. Both cell lines were approximately 10⁴ times more sensitive to 254 nm irradiation than to 365 nm radiation when irradiated aerobically. Pretreatment with 6 times 10⁵ Jm^-2 365 nm radiation sensitised the HeLa, but not the V-79 cell line to subsequent X-irradiation. Pretreatment of cells with 17 Jm^-2 254 nm radiation, a dose calculated to produce twenty times more pyrimidine dimers than the 365 nm dose, produced only slight sensitisa-tion to X-rays. It is suggested that the sensitisation to X-rays seen in the HeLa cells after 365 nm treatment is not the result of lesions induced in DNA by the near-UV radiation, but may reflect the disruption of DNA-repair systems.     

Induction of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Ultraviolet Radiation in Calf Thymus DNA and HeLa Cells

Abstract— The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8-oxodGuo was induced in purified DNA in a linear dose-dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8-oxodGuo per 10⁵ 2'-deoxyguanosine (dGuo) per kJ/m² for UVC, UVB and UVA, respectively. However, the amount of 8-oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8-oxodGuo per 10⁵ dGuo per kJ/m², respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8-oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV-induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8-oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC- or UVB-irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8-oxodGuo to dimers increased nearly 1000-fold in both UVA-irra-diated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8-oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.